Boundary-Free Ribosome Compartmentalization by Gene Expression on a Surface

• DOI: 10.1021/acssynbio.0c00613
• Publication Date: 2021-02-18T02:38:20Z
• Authors: Michael Levy; Reuven Falkovich; Ohad Vonshak; Dan Bracha; Alexandra M. Tayar; Yoshihiro Shimizu; Shirley S. Daube; Roy H. Bar-Ziv
• Keywords: 0301 basic medicine; Chemical Sciences not elsewhere classified; DNA brush layout; evanescent field fluorescence; Biophysics; 612; Biochemistry; Microbiology; Models, Biological; 2 D subcompartments; Inorganic Chemistry; 03 medical and health sciences; Boundary-Free Ribosome Compartmenta.; Dense DNA brushes; compartmentalize gene-expression re.; Genetics; RNA, Messenger; Molecular Biology; Cell-Free System; Computational Biology; Cell Biology; DNA; DNA-Directed RNA Polymerases; ribosome; Protein Biosynthesis; RNA; DNA brushes; protein; transcription; Ribosomes; Biotechnology; Biological Sciences not elsewhere classified; Fluorescence Recovery After Photobleaching

The design of artificial cell models based on minimal surface-bound transcription-translation reactions aims to mimic the compartmentalization facilitated by organelles and inner interfaces in living cells. Dense DNA brushes as localized sources of RNA and proteins serve as synthetic operons that have recently proven useful for the autonomous synthesis and assembly of cellular machines. Here, we studied ribosome compartmentalization in a minimal gene-expression reaction on a surface in contact with a macroscopic reservoir. We first observed the accumulation and colocalization of RNA polymerases, ribosomes, nascent RNAs and proteins, in dense DNA brushes using evanescent field fluorescence, showing transcription-translation coupling in the brush. Fluorescence recovery after photobleaching showed that ribosomes engaged in translation in the brush had a 4-fold slower diffusion constant. In addition, ribosomes in the brush had over a 10-fold higher local concentration relative to free ribosomes, creating a boundary-free functional ribosome-rich compartment. To decouple translation from transcription, we immobilized dense phases of ribosomes next to DNA brushes. We demonstrated that immobilized ribosomes were capable of protein synthesis, forming 2D subcompartments of active ribosome patterns induced and regulated by DNA brush layout of coding and inhibitory genes. Localizing additional molecular components on the surface will further compartmentalize gene-expression reactions.